3 Results and Discussion
3.1 Conserved architecture of the core RNA polymerase II initiation complex initiation complex
The following text in Section 3.1 was published.
Wolfgang Mühlbacher*, Sarah Sainsbury*, Matthias Hemann, Merle Hantsche, Franz Herzog, and Patrick Cramer. Conserved architecture of the core RNA polymerase IIinitiation complex.Nature comm. 2014;5:4310.
*These authors contributed equally.
Preparation and crosslinking analysis of the core ITC 3.1.1
To experimentally define the domain architecture of the core initiation complex, we reconstituted a defined yeast core ITC. We previously noted that a stable, defined ITC can be formed by including a 6 nt RNA product41. We therefore incubated purified Pol II, TFIIF, TFIIB, and TBP with a DNA-RNA scaffold (Figure 2a) and obtained a stable core ITC after size exclusion chromatography (Figure 2b) (see Online Methods). We then analysed this complex by XL-MS. The power and reliability of XL-MS was recently exemplified by a high agreement between Pol I models derived from XL-MS82 and subsequently from X-ray analysis91.
We obtained a total of 472 high-confidence lysine-lysine protein crosslinks (332 distance restraints) within the core ITC (Supplementary Table 1 and 2), of which 241 were inter-subunit and 231 were intra-subunit crosslinks (Table 8). A total of 194 crosslinks within Pol II were readily explained with the Pol II crystal structure92. Another 33 crosslinks were observed between TFIIF subunits Tfg1 and Tfg2, and
Most of these could be explained by the known structural flexibility and higher crystallographic B-factors of the involved lysine residues, leaving only three crosslinks unexplained. Within TFIIB and TBP, 23 and six intramolecular crosslinks were observed, respectively, and could be explained with crystal structures40,41,94,95
. These multiple internal controls demonstrate the high reliability of the observed crosslinking data.
Positions of TFIIB and TFIIF 3.1.2
We also observed 111 intermolecular crosslinks between transcription factors and Pol II (Table 8) that allowed us to model the core yeast ITC (Figure 3a). Of these crosslinks, 21 were observed between the TFIIF dimerization module and the Pol II lobe and protrusion domains, showing that the module remains at its location observed in the binary Pol II-TFIIF complex35,42. Further, 16 crosslinks were obtained between the TFIIB linker and the Pol II domains clamp core, protrusion, and wall.
Another six crosslinks were detected between the N-terminal TFIIB cyclin domain and the clamp core, protrusion and wall. The C-terminal cyclin domain did not crosslink to Pol II, consistent with its mobility40. All TFIIB-Pol II crosslinks were explained with our crystal structures of the Pol II-TFIIB complex40,41, demonstrating that TFIIB binds Pol II as observed in the binary structure. These results were corroborated by crosslinks between the TFIIF subunit Tfg2 to TBP (one crosslink) and to the N-terminal cyclin domain of TFIIB (six crosslinks).
The Tfg2 WH domain swings over DNA in the cleft 3.1.3
The crosslinking data also revealed that in the reconstituted core ITC the WH domain in Tfg2 can reside at a position near upstream DNA on the outside of Pol II42, but also at a position above the DNA in the active center cleft (Figure 3a, d). Thus, in the core ITC, the WH domain remains flexible and adopts both alternative positions. The original WH position near upstream DNA42 gives rise to 13 crosslinks to the Pol II subunits Rpb2, Rpb3, and Rpb10. The new position above the Pol II cleft was defined by four crosslinks of the WH domain to the clamp, TBP, and the TFIIB N-terminal cyclin domain. These restrains can be satisfied when one assumes a position of the WH domain with respect to promoter DNA that resembles that in a known X-ray structure of a WH domain bound to DNA96. This position is also consistent with a recent mapping of the DNA-binding face of the Tfg2 WH domain97.
These results indicate that the Tfg2 WH domain can swing over promoter DNA after its loading into the Pol II cleft, and indicate a role of this domain in DNA melting and/or stabilization of the open complex and the ITC. Indeed, this domain binds DNA and is required for initiation98, and TFIIF suppresses abortive initial transcription99. The proximity of the Tfg2 WH domain to TFIIB indicates how TFIIF could stabilize TFIIB on Pol II during initial transcription100. The position of the Tfg2 WH domain above the cleft apparently represents its position in a complete ITC. This position is near TFIIE and TFIIA in the PIC37,38, and is likely stabilized upon TFIIE and/or TFIIA binding. The other WH domain in TFIIF subunit Tfg1 only gave rise to a single crosslink at the Pol II jaw, and does not adopt a defined location42.
Model of the yeast core ITC 3.1.4
Based on the large number of protein crosslinks we built a reliable three-dimensional model of the yeast Pol II core ITC. First, we derived a homology model of the yeast TFIIF dimerization module based on the human crystal structure93. Second, we positioned the resulting yeast TFIIF dimerization module model onto the Pol II-TFIIB-DNA-RNA crystal structure41 assuming the location of the human module detected by EM37. Third, we extended DNA both upstream and downstream using standard B-form duplexes.
Figure 2: Preparation and XL-MS analysis of the yeast core ITC.
(A) DNA-RNA scaffold based on a HIS4 DNA promoter with a mismatched bubble region containing a 6 nt RNA transcript formi ng a hybrid duplex with the DNA template strand41. (B) SDS-PAGE analysis of the purified Pol II ITC revealing its 16 polypeptide subunits. (C) Cα distance distribution for observed lysine-lysine crosslink pairs (unique distance restraints). Crosslinks with distances of 30-39 Å are explainable due to protein mobility (four crosslinks) or because of lysine location in mobile protein loops with high crystallographic B-factors (14 crosslinks). Only three crosslinks cannot be explained and are classified as outliers. (D) Crosslink map of the ITC. Crosslinks within Pol II were excluded for clarity. TFIIB and Pol II subunits are colour-coded as before41 and TBP and TFIIF were coloured as in Figure 3. The map was generated with a MATLAB® script by coauthor Simon Neyer (see Supplementary Material 1).
TFIIF arm and charged helix 3.1.5
In the resulting model, the Tfg1 ‘arm’ (a β-hairpin comprising yeast residues 146-153 and 319-338) extends from the TFIIF dimerization module, traversing between the Pol II protrusion and lobe domains into the active center cleft (Figure 3c). The arm forms 19 crosslinks in the cleft, consistent with detection of the arm in the human PIC by EM37. A mutation at the point where the arm extends from the dimerization module leads to shifts in the transcription start site101.
A second extension from the TFIIF dimerization module, a negatively charged, flexible93 α-helix at the beginning of the ‘charged region’ in Tfg1 (named here the
‘charged helix’, yeast residues 406-417) clashed with the Pol II lobe. A reorientation of the charged helix towards the jaws released this clash and explained 6 crosslinks of the charged helix to the Pol II cleft. The location of the charged helix explained a distinct EM difference density that was hypothesized to stem from the corresponding human TFIIF region37.
Published mutational and kinetic data revealed important roles of the charged helix in transcription initiation and elongation102,103. These roles can now be rationalized due to the location near downstream DNA (Figure 3c). The charged helix apparently repels the downstream DNA from the lobe, positioning it along the clamp head on the opposite side of the cleft. This may help to stabilize melted DNA and to align the DNA template in the active site and account for the known role of the charged helix in stimulating initial RNA synthesis104.
Conclusion 3.1.6
Our crosslinking data and detailed architectural model of the core yeast ITC agree with previous site-specific protein cleavage mapping of the yeast PIC34,35,105. Our model further agrees with structural analysis of human Pol II PIC intermediates by EM37. Thus the architecture of the core ITC is highly conserved between yeast and human. Domains in TFIIF and TFIIB adopt very similar locations on the Pol II surface in both species, although the position of the Tfg2 WH domain above the cleft may change slightly upon binding TFIIA and/or TFIIE or different DNA template sequences. Furthermore, two TFIIF motifs extending from the dimerization module, the arm and charged helix, adopt the same locations in the downstream cleft. Our results also indicate that the overall domain architecture of the initiation complex is generally maintained during the transition from a PIC to an ITC.
Finally, our core ITC model can explain the XL-MS data obtained recently with a complete yeast PIC38. In the latter study, 117 distance restraints were obtained for Pol II, TFIIB, TFIIF and TBP. Of these, only one crosslink disagrees with our model, which was derived from 472 crosslinks with 332 distance restraints. Apparently the published study38 contains correct crosslink information, but conflicting electron microscopic results, which have apparently led to an alternative initiation complex model. With respect to the core ITC, the discrepancies are now resolved. Our results lead to a unified, highly conserved architecture of the core transcription initiation complex. The location of the remaining general transcription factors TFIIE and TFIIH differs to some extent in three published studies36-38 and may be analyzed in the future.
Table 8: Observed lysine-lysine crosslinks in the yeast core Pol II ITC.
Provided is the number of crosslinks between certain parts of the ITC, referring to unique distance restraints.
ITC parts Crosslinks
(all)
Crosslinks (mapable) All (inter and intra crosslinks) 472 328
inter crosslinks
All 241 164
Pol II-Pol II 90 90
Pol II-TFIIF 84 40 (48)1
Pol II-Tfg1/2 dimerization module2 25 (29) 25 (29)
Pol II-Tfg1 WH 1 1
Pol II-Tfg2 WH 14 (17) 14 (17)
Pol II-TFIIB 27 23
Tfg1-Tfg2 33 11
TFIIB-Tfg2 6 0 (1)
TBP-Tfg2 1 0 (1)
intra crosslinks
All 231 164
Pol II 104 104
TFIIF 98 32
Tfg1/2 dim.-module2 4 4
Tfg1 WH-model 18 18
Tfg2 WH-model 11 11
TFIIB 23 22
TBP 6 6
1Numbers in brackets include crosslinks that involve amino acids located no more than three residues away from residues within known structures.
2Residues from the charged region of Tfg142 (400-417) and N-terminal region (92-98) are also part of the dimerization model based on the human X-ray structure.93
Figure 3: Crosslinking-derived model of the yeast core ITC.
(A) Top view of the ITC, highlighting the locations of TBP (red), TFIIB (green), and TFIIF subunits Tfg1 (light blue) and Tfg2 (pink) on the Pol II surface. The TFIIF arm and charged helix elements are indicated as an antiparallel β-hairpin and α-helix, respectively. Alternative positions of the Tfg2 WH domain are indicated with black circled numbers (1, outside the cleft near upstream DNA as in the Pol II-TFIIF binary complex; 2, at the DNA bubble above the cleft). Mobile linkers are shown as dashed lines. (B) Pol II-TFIIB crosslinks (blue lines) viewed from the top as in (a) can be explained with the previously derived crystallographic TFIIB (B) core and ribbon domain locations40,41. (C) Location of the Pol II-TFIIF dimerization module (pink and yellow lines depict inter- and intra crosslinks, respectively). (D) The Tfg2 WH domain adopts two distinct locations. At position 1, the Tfg2 WH domain crosslinks to Pol II (pink lines), and at position 2, it crosslinks additionally to TFIIB and TBP (orange and red lines, respectively). (E) Domain organization of TFIIF subunit Tfg1 and location and conservation of the arm and charged helix elements. The charged helix was partially resolved in the X-ray structure93 and is predicted to be longer106. Residues required for normal transcription initiation and elongation102 are indicated as grey asterisks.